Gene Activated Matrices for Bone and Cartilage Regeneration in Arthritis

Final Report Summary - GAMBA (Gene Activated Matrices for Bone and Cartilage Regeneration in Arthritis)

Executive Summary:

The GAMBA consortium has aimed at creating a novel gene-activated matrix platform for bone and cartilage repair with a focus on osteoarthritis-related tissue damage. The S&T objectives of this project have been complemented with an innovative program of public outreach, actively linking patients and society to the evolvement of this project. The GAMBA platform has been conceived to implement a concept of spatiotemporal control of regenerative bioactivity on command and demand. The platform comprises modules that can be independently addressed with endogenous biological and exogenous physical or pharmacological stimuli, resulting in a temporally and spatially coordinated growth factor gene expression pattern. This has been intended to reproduce key elements of natural tissue formation. The modules are growth factor-encoding gene vectors, mesenchymal stem cells, magnetic nanoparticles, a ceramic matrix and a biomimetic hyaluronan gel. Anatomical adaptivity was to be achieved with engineered thermal properties of the hyaluronan matrix, which embeds the other modules, selected according to functional requirements. Mechanical support was to be provided by the Micro Macroporous Biphasic Calcium Phosphate (MBCP™), a resorbable material approved for clinical use. Spatiotemporal control of bioactivity and responsiveness to physiological conditions was to be represented, firstly, in the spatial distribution and release profiles of gene vectors within the composite matrix and, secondly, by letting local and external biological or physical stimuli activate the promoters driving the expression of vector-encoded transgenes.

The project had four major objectives: 1) Establishing GAMBA, creating the construction kit – matching materials science and gene vector development to achieve hierarchies of spatiotemporal control of bioactivity on command and demand; 2) Validating GAMBA – matching biophysics and cell biology in order provide proof of concept in cell culture models and eventually in animal models; 3) Linking GAMBA to society through innovative outreach methods including teaching students, discussing chances and risks in patient and citizen panels and raising awareness for gender equality within and outside the consortium; 4) Disseminating and exploiting project results through publications, public relations efforts and eventually through commercial product developments. To achieve these objectives, the project was structured in 7 scientific workpackages complemented by the outreach workpackage. Two further workpackages were dedicated to dissemination / exploitation and project management. The essential goals of the project have been reached. In a first step, all required vector constructs were established and distributed among the partners. Inducible gene expression was demonstrated in cell lines. However it turned out that some of the inducible systems did not work equally well in mesenchymal stem cells which are an essential element of the GAMBA platform. Hence, only those constructs could be used that did work in this cell type. While some of the GAMBA partners continued their basic research on the biology of bone and cartilage regeneration, notably with patient cells, in order to define the preconditions for the GAMBA platform to work, the vector and biomaterial engineers within the project started to assemble the modules to demonstrate spatial and temporal control of reporter gene expression in 3-dimensional setups of the GAMBA modules. The individual engineering tasks proceeded quite successfully with reporter genes. It turned out much more challenging to demonstrate cartilage (re)generation and anti-inflammatory action with the individual genes that had been chosen beforehand based on the scientific literature while the induction of bone formation was demonstrated successfully in vitro and in vivo. Achieving spatio-temporal control with three different therapeutically relevant genes remains to be accomplished.

The GAMBA project has successfully created a toolbox of individual elements that will be used in follow-up projects. Possibly the most successful and exciting part of the project was public outreach in patient and citizen panels which was greatly appreciated by the participating lay persons and scientists alike. As part of the GAMBA project, 20 papers were published and 9 master and PhD theses were completed. Numerous press articles and broadcasts made the wider public aware of the project. Three commercial gene delivery products for tissue engineering were launched and one patent application is under way. Summarizing, this project is a success upon which future generations of follow-up projects can build on.

Project Context and Objectives:

Context and objectives

Due to demographic and life style changes, degenerative diseases are an enormous medical and socio-economic challenge in industrialised nations. Among them, the musculoskeletal diseases osteoarthritis (OA), rheumatoid arthritis and osteoporosis are the most prevalent. There is currently no gold standard for the repair or prevention of onset of OA. There are three major challenges: reducing inflammation, cartilage repair and subchondral bone repair. Biomimetic approaches for tissue repair in osteoarthritis require tight spatiotemporal control of bioactivity in order to address these challenges in a coordinated fashion.

No single established treatment of tissue defects – including OA - implements the features of natural tissue formation, namely cell differentiation in response to spatiotemporally controlled growth factor gene expression patterns on command and demand. GAMBA was conceived to implement these features and has focussed on innovative materials that induce and sustain bone and cartilage healing against the background of osteoarthritis. Osteoarthritis (OA) is a degenerative disease of the joints affecting, above the age of 45, more women than men, with an incidence increasing with age. It is estimated that 80 % of the population will have radiographic evidence of OA by age 65. The cartilage of the affected joint becomes rough and degenerates. With disease progression, the cartilage disappears and bone rubs on bone. The population burden of OA will increase over the next years due to the ageing population and the rising prevalence of obesity, being the principal non-genetic risk factor for OA. This leads to tremendous economic burden. The etiology of OA is still unknown. The current management of osteoarthritis is not regenerative but merely symptomatic, aimed at reduction of pain, controlling inflammation with non-steroidal anti-inflammatory drugs with an ultimate option of total joint replacement.

Healthy tissue features unique plasticity characterised by continuous remodelling in response to physiological and external stimuli, resulting in a controlled balance of anabolic and catabolic processes. Tissue damage is characterised by imbalance, loss of control and in the case of osteoarthritis a dominance of catabolic processes.

Innovative therapeutic concepts are regenerative in nature, thriving on the regenerative potential imprinted in our genetic background. The challenge is seizing this potential. This can be achieved, in principle, with growth factors and stem cells. While the latter are inherently multipotent, the former can be used to reawaken silenced endogenous programs of tissue formation, in a similar way to the spatiotemporal concentration gradient of morphogenesis observed during embryogenesis. These programs consist initially of temporally and spatially coordinated gene expression patterns, leading ultimately to a spatially and temporally concerted action of growth factors. In the context of tissue regeneration with biomimetic implants, such concerted action is exceedingly challenging to reproduce with recombinant growth factors as they have no inherent elements for responding to physiological conditions or external stimuli. In contrast, gene vectors can be engineered to have these elements and thus can reproduce spatiotemporally controlled gene expression patterns in situ.

Hence, the main objective of the GAMBA project was developing a biomimetic implant system using a nanobiotechnological approach that delivers regenerative bioactivity in a temporally and spatially controlled fashion in response to endogenous and external stimuli. In this manner, the system was designed to respond to and control inflammation and induce cartilage and subchondral bone repair in OA-related tissue defects. The implant system was designed alter the phenotype of cells in a physiologically meaningful way to elicit the desired therapeutic outcome.

For this purpose, the GAMBA consortium has aimed at creating a novel gene-activated matrix platform for bone and cartilage repair with a focus on osteoarthritis-related tissue damage. Three therapeutically relevant genes under the control of independently inducible promoters were to be vectorized and co-incorporated with mesenchymal stem cells in a 3-dimensional compartmentalized matrix. The compartments were to mimic the situation in an osteochondral bone defect, namely damaged bone and cartilage to be regenerated while the cartilage facing an inflammatory destructive environment. Hence, there was to be a vectorized gene encoding an osteoinductive factor in the first compartment, another one encoding a cartilage-inducing second compartment and a third one encoding an anti-inflammatory factor in the third compartment. The compartmentalization was to be realized with bone replacement material (MBCP™, a resorbable material approved for clinical use) impregnated with the bone-inducing vector and co-incorporated with the stem cells in a hydrogel matrix that in the upper layer would comprise the cartilage inducing and inflammatory vectors. While residing in the compartmentalized matrix, the stem cells would be transfected, start to express the therapeutic genes and in consequence differentiate to bone and cartilage cells while also fighting inflammation by producing the anti-inflammatory factor. To make the system controllable in a temporal fashion, the three therapeutic genes were to be switchable by independently inducible promoters. This scientific concept was to be complemented with an innovative program of public outreach, actively linking patients and society to the evolvement of this project.

Concequently, the project had the following objectives:

Objective 1: Establishing GAMBA, creating the construction kit – matching materials science and gene vector development to achieve hierarchies of spatiotemporal control of bioactivity on command and demand

1. Create spatiotemporal control elements based on biological/pharmacological principles with nonviral and adenoviral vectors with feedback response elements driving transgene expression. Vectors were to be constructed such that inducible promoters drive transgene expression: Vectors responding to inflammatory signals with the expression of anti-inflammatory factors (Cox-2 promoter). Vectors responding to temperature (HSP70 promoter driving growth factor gene expression; induction by AC magnetic field hyperthermia). Vectors responding to doxycycline (tet-on system driving the growth factor gene expression).
2. Create spatiotemporal control elements based on physical principles. A thermo-responsive hyaluronan-based biomimetic polymer and embedded therein MBCP™, a calcium phosphate ceramic, were to be used as carriers for gene vectors. Magnetic nanoparticles were to be co-embedded. Spatial control was to be achieved by differential vector positioning within the composite. Spatiotemporal control was to be demonstrated based on vector release patterns including thermally induced release generated by AC magnetic field hyperthermia and magnetic nanoparticles.

Objective 2: Validating GAMBA – matching biophysics and cell biology

1. Demonstrate spatiotemporal control of transgene expression with selected vector/biomaterial combinations in cell and tissue culture models, by evaluating transduction/transfection efficiencies, transgene expression duration and effectiveness of growth factor secretion with time. As control elements we will use differential vector release, molecular biological / pharmacological means (doxycycline), physical (heat) and molecular-biological (HSP70) means.
2. Obtain anti-inflammatory response in vitro with gene activated compound materials encoding IL-10 under the control of Cox-2 promoter. The Cox-2 promoter responds to inflammatory cytokines and thereby provides a feedback control element for the expression of the anti-inflammatory cytokine IL-10. This process was to be characterised in vitro, ex vivo and finally in a small animal model.
3. Using the GAMBA platform to obtain spatiotemporally controlled chondrogenesis or osteogenesis with MSCs embedded in gene activated compound materials. For induction of chondrogenic differentiation TGF-ß expression and for stimulation of osteogenesis BMP-2 expression was to be induced by promoters that respond to doxycycline or heat to be able to regulate location, time and duration of gene expression. Assessment was to be performed in vitro, ex vivo and in suitable small animal models of osteochondral defects.
4. Obtain proof of concept for GAMBA.

To finally ascertain the success of GAMBA, a large animal model (goat) was to be used. The choice of the model will depend on the relative successes of the previous objectives with an osteochondral defect being the model of choice assuming the success of all previous objectives.

Objective 3: Linking GAMBA to society through innovative outreach methods

1. Educating students in nanomedicine. Each Partner currently participates in local education programs for undergraduate students. We will incorporate the GAMBA concept in these existing courses as well as attract undergraduate and graduate students to participate in the project.
2. Integrate patients (esp. female/elderly) into the development of innovative treatments. This was to be accomplished with so-called Patient Panels. OA patients were to be invited to participate in workshops together with the scientists carrying out this project and were to be asked to communicate their needs and views in relation to this project. The patients will compile their expectations and recommendations on regenerative medicine in writing. Proceedings from the patient panels were to be made available to scientists, the interested public, regulators and politicians via an internet discussion forum and other PR measures (see chapter 3.2) together with results from (3) below.
3. Discuss chances, risks and ethical aspects of GAMBA with the general public. In a format similar to the Patient Panels, Citizen Panels were to be held in order to enhance awareness of nanomedicine in the public and take into account public expectations and reservations. Randomly selected citizens were to be invited to discuss the chances, risks and ethical aspects of GAMBA together with experts from different scientific disciplines facilitated by neutral communication experts. The participants were to compile a Citizen Report with recommendations on regenerative medicine that were to be published and disseminated to interested scientists, medical personnel, regulators, the media and the interested general public.
4. Raise awareness for and discuss gender equality. A Partner workshop was to be organised to raise awareness for gender issues within the Partner organisations and to discuss expectations and needs of female employees regarding gender equality. The goal of this workshop is to establish the professional networks for female project members that are essential for a sustained professional career. Achieving this goal is facilitated by the fact that 4 out of 8 of the Principal Investigators in this project (including the subcontractor PIs), among them notably the coordinator, are female.

Objective 4: Disseminating and exploiting GAMBA

1. Transfer of project results into research and clinics through the participating companies. Three of the Partners are located in academic hospitals and are in close contact with patients. Medical doctors were to be actively involved in the project from an early stage. The companies involved will guarantee protection of IP rights. We will organise specific meetings with medical specialist and business people to discuss the needs and possibilities to get our idea into a clinical application. There was to be at least one such meeting at the start, one at midpoint and one at the end of the project. Stakeholders will also be invited to participate in Patient Panels and Citizen Panels of Objective 3.
2. Public relations campaign. The presence of members of international/national associations in regenerative medicine within the consortium will significantly enhance the possibility to disseminate new data to as wide an audience as possible. After the IP potential has been evaluated and secured, data and results were to be made public to the scientific and wider community. As the project is progressing the new information was to be disseminated.

Project Results:

WP 1: Vector development for spatiotemporally controlled gene expression

Lead beneficiary: TUM

WP leader: Christian Plank, Martina Anton

The aim of this WP was to construct and characterize gene vectors suitable to independently regulate gene expression of reporter genes or therapeutically active genes that is to say genes that are known to be involved in regeneration of cartilage or bone or in reducing inflammation in joints. The aim was to produce non-viral and viral vectors, optimize them for transduction efficiency, show their functionality in cell culture experiments and prepare and analyze assemblies of gene vectors and scaffolds with cells.

TUM successfully constructed a series of inducible plasmid and adenoviral gene vectors combining as regulatory elements either the TET-on system, a heat-inducible Hsp70B based promoter or an inflammation inducible Cox-2 promoter. These were used to drive expression of bone morphogenetic protein 2 (BMP-2), transforming growth factor beta1 (TGFb1 or viral Interleukin 10 (vIL10), which was chosen, since vIL10 was reported to exhibit solely anti-inflammatory activity. Additionally gene vectors carrying the respective cDNAs under control of constitutively active promoters were produced as controls.

These vectors were analyzed for their functionality after infecting or transfecting tumor cells, mesenchymal stem cells or chondrocytes and by applying the respective stimulus. Heat induction was by simple incubation at elevated temperatures and turned out to be optimal at 43°C; inflammation was mimicked by either applying LPS and TPS or IFNgamma and TNFalpha and the choice of inducers was dependent on cell type; pharmacological stimulation was by addition of doxycycline (DOX) to tissue culture medium. Induction was demonstrated by reporter gene assays, fluorescence microscopy and flow cytometry and by ELISA for secretion of the growth factors and the cytokine. All regulation systems showed inducibility by the respective stimulus in tumor cell lines. However induction levels of the heat-inducible vectors was low as well as for the inflammation inducible Cox-2 promoter. Additionally no inducibility of both promoters was seen in MSC either due to toxicity of non-viral vectors with heat or background activity of the Cox-2 vectors in hMSC (NUI Galway, EMC). Production of therapeutic proteins was expected not to reach critical levels that might drive differentiation of MSC. In contrast, the TET-on system resulted in high induction levels for reporter genes and BMP-2 in different cells, most importantly in MSC, with only background expression in the absence of inducer. These highly favourable characteristics lead to construction of new vectors for TET-on vIL10 expression, which resulted in tightly regulated and high vIL10 expression after infection of rMSC. Since results of WP5 had shown that TGFb needs to be expressed for extended periods of time, which cannot be achieved by heat shock the continuously active CMV promoter was chosen.

This way the ability of the vector systems for temporal gene expression was clearly demonstrated for all induction systems tested, but the choice of promoters needed to be adapted to the expression times and levels needed for potential biasing MSC differentiation.

Due to their higher expression levels, adenoviral vectors were chosen for further analysis for functionality of produced therapeutic proteins. The influence of switched “on” (and “off”) gene expression of the growth factors BMP-2 and TGFb and the cytokine vIL10 on osteogenic and chondrogenic differentiation of MSC was analyzed. Rat MSC infected with different Dox-inducible and constitutively expressing adenoviral vectors were subjected to standard in vitro differentiation assay for osteogenesis (Ca2+ quantification and alizarin red staining) and pellet mass assays for osteogenic differentiation (glycosaminoglycan quantification and alcian blue staining). At the same time the level of therapeutic gene expression was monitored over time and in the case of constitutive gene expression turned out to be decreased as expected, either due to loss of vector (non-integrating systems) or due to epigenetic silencing. Under differentiation conditions DOX-inducibility was retained. At early time points high induction levels were achieved, but DOX-induced level of therapeutic proteins decreased over time although induction was still possible at the end of differentiation assays. Experiments revealed that the Tet-induced BMP-2 expression enhances osteogenic and to a lesser extent chondrogenic differentiation of MSC. Non-regulated TGFb1 enhances chondrogenic differentiation, although being produced at a low level, thus replacing recombinant TGFb protein. vIL10 overproduction had a negative impact on osteogenic differentiation, but did not influence chondrogenic differentiation negatively. Thus with the Tet-induced system we could successfully demonstrate temporal control of growth factor expression and showed the functionality of all therapeutic gene vectors in a 2D model of chondrogenesis and osteogenesis.

Spatial control of gene expression was shown by assembly of modules (OZB) with reporter genes using different scaffolds for gene vectors and cells provided from partners (ARI, BIM, INSERM) or commercially available gels. Spatially differential expression of reporter genes was seen using red and green fluorescence protein reporters (TUM), but were difficult to quantify and demonstrate unequivocally using the thermosensitive hyaluronan gel (ARI).

With 3D-assembly of different scaffolds, vectors and cells TUM was able to confirm temporally regulated BMP-2 expression combined with non-regulated TGFb expression. So far using qRT-PCR assays of MSC in/on the different scaffolds did not demonstrate any influence on differentiation by the therapeutic proteins produced due to low cell numbers. These experiments need optimization and are still ongoing. However evading cells hinted at increased Ca2+-deposition under Doxycycline addition and Tet-regulated BMP-2 expression was increased. It was demonstrated by ELISA, that even in 3D- culture the Tet-on system is functional for BMP-2 expression and that Dox has no negative influence on the constitutive TGFb expression in the same assemblies.

As an alternative to demonstrate spatial control of gene expression, a 2D bioluminescence imaging system was chosen by TUM. Here one of the inducers of the Cox-2 promoter was locally produced, secreted into the medium and after diffusion resulted in induction of Cox-2 promoter-driven luciferase bioluminescence of cells seeded in a different locus of the same dish.

Thus within the GAMBA project the concept of spatial and temporal control of gene expression was successfully demonstrated.

Additional tasks of WP1 dealt with optimizing gene vectors by shielding and embedding of gene vectors into/onto scaffolds and magnetic gene vectors and magnetofection.

On the nonviral side, a high throughput screening of a lipid library by OZB led to the selection of two DNA transfection candidates for both 3D-hydrogels and 3D-Scaffolds. These two compounds were optimized on a variety of matrices and were later commercially launched (3D-Fect™ & 3D-FectIN™). Thereafter, new lipids and polymers were further synthesized to solve the transfection problems associated with the combination of thermo-responsive HA-PNIPAM gel with the hydrophobic lipids. New lipids and polymers candidates with reduced hydrophobic properties were synthesized, selected and sent to partners 1, 2 and 7 (MRI, ARI and IRCCS). Physical and chemical characterization and biological evaluation were performed for these compounds (see WP2 ).

OZB has then selected the best formulation to transfect chondrocytes and hMSC in classical cell culture conditions (before implantation in the biomaterials) (1) with high efficiency, (2) low toxicity and (3) without inducing any visible phenotypic changes. The lipids DOGTOR and NL-37 induced highest transfection efficiency in terms of number of cells transfected and transgene expression. Moreover, CombiMag (Magnetofection) allowed raising the transfection efficiency of both reagents. Importantly OZB also defined critical parameters that influence the transfection efficiency including cell density, culture conditions, passage number, days of transfection, medium change etc. OZB has then produced a detailed protocol to standardize the transfection procedure of hMSC that was disseminated with the reagents to all partners. DOGTOR was finally adopted by all partners to be the reagent of choice for transfecting stem cells in 2D.

With respect to vector shielding, the partners had pre-existing knowledge and compounds ready. Polymers and/or magnetic nanoparticles (MNP) and were analysed for their physical properties, including release from scaffolds and gene expression levels. Surprisingly, magnetic nanoparticles alone exerted an excellent surface shielding effect on the adenoviral vector which was at the same time transfection enhancing when the vector was co-incorporated into 3D culture format together with cells.

The generation of magnetic particle-associated vectors was an important task in the project also for another reason. One modality of the induction of transgene expression was via heat generation by AC magnetic field action on magnetic nanoparticles. This required a high concentration of the particles in the 3D phase. And luckily this turned out to enhance adenoviral transduction, as mentioned. From a collection of MNP, two types were identified by TUM that resulted in a satisfying increase of temperature under AC magnetic field. Furthermore, these particles were compatible with the gelation properties of the hyaluronan-based hydrogel (ARI, TUM). These particles were finally used to induce luciferase expression by AC magnetic field induction in a cell line that carries the gene under control of a heat-inducible promoter. The system still needs improvement, but proof of principle was achieved. As AC-MF instruments are not widely available, a swift technology transfer to other partners was not possible.

As a side-product of research in nanomagnetic vectors, OZB developed and published the i-MICST method for integrated cell sorting and transduction of MSC with adenoviral vectors. This method is highly efficient in generating genetically modified mesenchymal stem cells and can be used advantageously for certain objectives of the GAMBA project.

Conclusion:

In WP1 we successfully constructed regulated non-viral and adenoviral gene vectors and demonstrated functionality with respect to regulation (TUM). These vectors were made available to all partners.

Non-viral and viral magnetic gene vectors have been successfully produced. Two compounds were optimized by OZB on a variety of matrices and were later commercially launched (3D-Fect™ & 3D-FectIN™). A detailed protocol to standardize the non-viral transfection procedure of hMSC was disseminated with the reagents to all partners. DOGTOR was finally adopted by all partners to be the reagent of choice for transfecting stem cells in 2D. Likewise the AC/MF heat induction had been investigated and demonstrated, but needs improvement. As AC-MF instruments are not widely available a swift translation to other partners was not possible.

Some of the regulatory elements (Cox-2- and Hsp70B promoters) turned out to be functional, but too weak to be expected to work in driving differentiation of MSC. Therefore the following alternatives were chosen, that allowed for stronger or constitutive expression: Tet-system for the regulated expression if vIL10 and the constitutive CMV promoter for TGFb expression.

With the Tet-induced BMP-2 system we could successfully demonstrate temporal control of growth factor expression and showed the functionality of all therapeutic gene vectors in a 2D model of chondrogenesis and osteogenesis. TGFb1 enhanced chondrogenic differentiation

The 3D-assembly of different scaffolds, vectors and cells was successful. Although we were not able to demonstrate bias of differentiation by gene vectors within the 3D assemblies due to low cell numbers, we were able to demonstrate that switching on of BMP-2 expression was successful in 3D as well as 2D culture. Thus the temporal control of gene expression by the Tet-on system was successfully implemented in 3D culture as well.

Spatial control of gene expression was demonstrated by reporter gene imaging and unequivocally in an independent system of luciferase imaging that relies on the local production of TNFalpha, which leads to Cox-2 promoter mediated luciferase expression in a spatially separate locus.

WP2: Development of a biomimetic polymer as carrier of gene vectors

Lead beneficiary: ARI

WP leader: Mauro Alini

Thus, the major objective of the workpackage 2 was to create a biomimetic polymer based carrier platform that allowed spatiotemporal control of cell behaviour using a combination of technological breakthroughs achieved by Partners of the consortium. The specific technical module developed in the workpackage 2 was a temperature-sensitive hyaluronan (HA) hydrogel and its combination with MBCP™ tailor-made granules (Micro macroporous Biphasic Calcium Phosphate), nanosized nonviral or viral gene vectors encoding desired growth factors under the control of inducible promoters, superparamagnetic nanoparticles and mesenchymal stem cells. All the partners were involved in this major workpackage.

The biomimetic hydrogel carrier developed in this project was based on hyaluronan which has important roles in organ development and cell signaling (Leach et al., 2004). Commonly, hyaluronan hydrogels are obtained by cross-linking reactions involving chemicals and/or UV light (Lapcik et al., 1998), which are potentially detrimental to biologics to be embedded in the gels. In the workpackage 2, the partner 2 (AOF) performed the grafting of thermo-responsive linear polymers (poly(N-isopropylacrylamide)) onto the hyaluronan either via Cu(I) catalyzed alkyne-azide cycloaddition (CuAAC) reaction or via direct amidation. The materials were characterized with notably; 1H nuclear magnetic resonance, thermal analysis, rheology, and swelling-shrinkage measurements, as well as stability test. The materials were successfully synthesized and showed a gel point in aqueous solution at 30-32•C. The grafting via amidation reaction did not lead to degradation of hayluronan as observed in the CuAAC reaction performed in the presence of the catalyst; CuSO4, 5H2O with sodium ascorbic acid salt. This leaded to improve reproducibility and potential scaling-up. Moreover, the absence of potentially toxic copper traces in the amidation reaction product increased the likeliness for use in vivo. The thermoresponsiveness of the hydrogels and the rheological features of the gels were modulated from room temperature to above body temperature depending on composition and concentration, hence providing temperature-dependent spatiotemporal control of the material properties. In the meantime, a biomimetic hyaluronan hydrogel platform based on a thiol-ene reaction crosslinking was developed in order to compare with the purely physical crosslinked hyaluronan carrier. Biomimetic functionalities, such as RGD binding epitope were easily added in this 2nd hyaluronan platform, enhancing hMSCs spreading in 3D hydrogel matrix. Preliminary cell study was performed in order to assess the viability of cells dispersed in a thermoresponsive hyaluronan composition after injection through a needle. Cell viability at 24 h after injection was 90 ± 2% for the thermoresponsive hydrogel compared to 71 ± 9% for alginate hydrogel control, confirming that the thermoresponsive hydrogel can be used as an injectable cell carrier. Further study on the encapsulation of human mesenchymal stem cells (hMSCs) has been performed by several partners showing that hMSCs viability at 7 days was higher or identical in the thermoresponsive hydrogel compared to alginate control hydrogel. Further, cell culture demonstrated the chondrogenic potential of the thermoresponsive hydrogel in vitro.

After, the optimization of the biomimetic hydrogels platform, combination with micro and macroporous biphasic calcium phosphate (MBCPTM and MBCP+TM) developed and produced by Biomatlante (BIM) was performed in order to create the osteochondral GAM. The MBCPTM was first considered to be the gene vector carrier encoding a first factor which will then be embedded in the hydrogel which acted as a cohesion enhancer for the MBCPTM particles, cells carrier or 2nd gene vector carried. In a whole, the composite GAM concept provided an element of spatiotemporal control in this project. The combination of MBCPTM granules of different composition and varying concentration with the thermo-responsive hydrogel solutions was performed in collaboration between AOF, BIM and INSERM partners. The preparation of composite materials was successful. It was found that the loading of the hydrogel solution with a quantity of particles up to eight time higher in weight of the polymer in solution, 1) increased the viscosity of the composition at temperature below the lower critical solution temperature (LCST), 2) decreased the LCST with the amount of granules added, 3) conserved the gelling property of the hydrogel and 4) increased the final storage modulus with the granule content. Full characterization of the composites was performed ranging from IR spectroscopy, rheology, X-ray diffraction, electron microscopy, etc. The stability of the formulations was evaluated observing the settling of the particles. Focusing on the handling of the composite matrices prepared extrusion force was measured on an Instron 4302 electromechanical testing machine equipped with a customized load cell. The different formulations were characterized by different stability depending on the particle size. It was seen that particle of size above 200µm tend to settle. Such compositions needed to be formulated at higher viscosity (for instance, addition of underivatized hyaluronan) at room temperature. All the compositions were extrudible at room temperature through a syringe. They had therefore suitable handling for use in a surgical theater.

Additionally, superparamagnetic nanoparticles have been combined with the biomimetic hydrogel platform: Magnetic nanoparticles are widely used in biology, bioengineering, bioseparation, medical diagnostics as well as in preclinical and early clinical studies with innovative therapies. The hydrodynamic diameters of the particles used were of few nanometers (about <10-20 nm) to micrometers in size. The particles prepared by partner 1, TUM, were made of iron oxide cores (magnetite or maghemite) and coated with biocompatible polymers which protects the core from degradation, stabilizes the particles in suspension and functionalises them according to the specific needs of biomedical applications. This resulted in physical properties which was deem suitable for the GAMBA project and application for a heat induced gene expression in a GAM carrier. Work performed by Partner EMC had demonstrated that iron oxide particles did not adversely affect cell behaviour. Thus, superparamagnetic nanoparticles have been prepared with specific surface charges in order to assess the influence of electrostatic interaction and therefore the stability of the hydrogel platform loaded with the particles. Hydrogel solutions containing the superparamagnetic nanoparticles, showed that negatively charge magnetic nanoparticles were more stable in solution at room temperature and that the gelling properties of the composition was conserved; constants LCST and Elastic modulus at 37•C. In the gel state above 32 •C stable dispersions were achieved. Remote thermal switching of functional states and release properties of the hydrogel embedded superparamagnetic nanoparticles were assessed using AC magnetic field induction. Temperature increase up to 43•C was achieved in such composite materials which would be enough to trigger the heatshock promoter able to switch on desired protein expression of gene transfect cells in a temporal fashion. This was confirmed by in vitro experiments performed by partner 1, TUM (WP1).

In the context of GAM combination with non-viral vectors, OZBioscience (OZB) studied the efficiency of lipoplexes to transfect hMSCs in various 3D hydrogel models such as atelocollagen, and biomimetic hyaluronic hydrogels. A lower efficiency of transfection into the thermoresponsive hyaluronan gel was hypothesized to be related to the hydrophobic interaction of lipids with the gel formation (thermoresponsive poly(N-isopropylacrylamide)). The exploration of the combination of lipoplexes or nanoparticles and thermoresponsive hyaluronan gel were followed in order to transfect cells in the biomimetic platform. No strategy (lipids, polymers, nanoparticles) was found to be effective in transfecting hMSCs into thermoresponsive hyaluronan gel. Physical and chemical characterization of the complexes (Magnetofection based reagent and lipid based reagents) was initiated by OZB and ARI to study the correlation between compatibility with thermoresponsive gel and complexes size and zeta potential. Size and zeta potential of complexes in different mediums are some of the parameters were analyzed to determine the best conditions for complete compatibility between transfection reagents and hydrogels. In parallel, newly synthesized HA hydrogel prepared by ARI was tested for its capacity to retain lipoplexes. Results showed that a high concentration of HA (>10%) was required to maintain the complexes into the gel. However under this condition, the hydrogel was difficult to handle. Consequently, OZB produced new lipids and polymers with minimized hydrophobic properties. The new compounds were effective in transfecting cells in 3D hydrogel made of atelocollagen but much less effective with hyaluronan gel. In order to gain a better insight on the physicochemical characterization of the complexes, OZB produced fluorescently labeled transfection reagents for studying the capacity of fluorescent complexes to be maintained inside the matrices according to the scaffold composition. The study allowed to gaining insights on several points: such as the different non-viral vector retention capacity of matrices. A clear outcome was that the embedding MBCP and MBCP+ granules, into hydrogel was necessary to retain the complexes inside the biomaterials and efficiently transfect colonizing cells. These results were in close relationship to the observed cell transfection data. These observations were of high importance for in vivo experiments as GAM bearing complexes must remain active several days to allow colonization and transfection of cells. OZB and ISTGE collaborated on in vivo mouse model and lipids non-viral vector newly synthesized and new GAM protocol. Preliminary results showed that fluorescence expression of transfect cells can be observed in the implanted scaffold. In addition to the above reported in vivo study, ARI has demonstrated that viral transfection of hMSCs in thermoresponsive hydrogel was highly efficient and that selected biomimetic hydrogel composition was biocompatibility (see WP7). For this last task, sterilization methods were screened, material batch synthesis was scaled up and assessed, and in vivo study performed.

To conclude, although high efficacy of non-viral vector into the thermoresponsive hydrogel matrix could not be reached, the GAM developed by the GAMBA team, as a combination of biomaterials and a biomimetic polymer, has reached a level where it can be used as gene vectors and cells carriers for pre-clinical studies.

WP3: Biomimetic Calcium Phosphate Bioceramics granules as carriers for gene vectors

Lead beneficiary: BIM

WP leader: Pascal Borget/Guy Daculsi

The concept of scaffold for the invasion of newly formed tissues at the expense of the latter is the cornerstone of bone tissue engineering. The challenge of the European project Gamba to combine gene therapy and bone tissue engineering involves optimizing bioactive materials supporting the reconstruction thanks to adhesion of host cells and vectors for gene transfection. Biomatlante company, synthetic bone substitutes manufacturer, and LIOAD Inserm laboratory of Nantes University, developed new formulations of bioactive ceramics to achieve objectives. Research and Development tasks necessary for the completion of this project are the scientific and technical heart. Biocompatibility tests were then performed in vitro and specific in vivo animal models have been set up to better meet the specifications and accurately assess the consistency of established biologics solutions. At maturity of 3 years of Gamba project, Biomatlante company and Inserm LIOAD laboratory were able to demonstrate unequivocally the effectiveness of ceramic scaffolds (MBCP+®) in terms of cells or transfection vectors adhesion, differentiation and bone ingrowth even in the most adverse situations (osteonecrosis).

I. Scaffolds optimization and new galenic formulations development

The demand placed on biomaterials for bone regeneration involves a total control of crystallographic, chemical and mechanical properties. Biological responses to these alloplastic grafts depend on these multiple factors that cannot be left to chance.

That is why Biomatlante company in collaboration with the laboratory Inserm LIOAD deeply characterized its newly developed medical devices for Gamba project in order to anticipate their positive bioactivity.

1) MBCP® technology (granules or blocks) [Task T3.1]

Bone knowledge is the base of biomimetic approach which leads to improve ceramic biomaterial synthesis approaching native bone in terms of chemical elements through the crystalline phase named hydroxyapatite. Biological apatite is form as nanocrystals after precipitation process. The materials engineering joining biomimetic and chemical strategies was used to develop methods to combine intimately crystalline phase of hydroxyapatite with called Beta-Tricalcium Phosphate (TCP), the latter being absent from the body. Resorption properties of TCP give the biomaterial, biphasic (HA / TCP), a "smart design". Indeed, the synthesis process control at high temperatures and the addition of porogens, allow offering dual-porosity, microscopic and macroscopic to these biphasic bioceramics with crystallographic grains size monitoring. Crystal size will influence microporosity, resorption and mechanical resistance. From this expertise was then created the MBCP concept, “M” for Macro-Microporosity, and “BCP” for Biphasic Calcium Phosphate.

2) New granules formulations with lower density and internal large concavity [Task T3.1]

As genic therapy and bone tissue engineering statements of Gamba project focused on host cells adhesion on osteoconductive scaffolds, Biomatlante and Inserm laboratory have invented a new galenic formulation of granules. These granules have been specifically design for several reasons:

- To protect cells from tribological forces between granules surfaces, which could damage cells on convexical external surfaces.
- To improve cell adhesion in a large internal concavity acting as a favorable osteopromotive calcium phosphate releasing “niche”.
- To decrease density of scaffolds which increases the resorption rate thanks to thin microporous walls and enhances the fluids penetrations. In other terms, with a lower mass of granules, you could obtain at least as much new bone volume than with high density granules.

These new granules are currently being patented by Biomatlante and Inserm LIOAD laboratory together.

3) Composite Ceramic/Hydrogel [Task T3.2]

Cohesion between hydrogel made of either hyaluronic acid (AO foundation, Davos, Switzerland) and ceramic granules (Biomatlante) have been tested, as well as chemical event monitoring after association with transfection vectors.

Two formulations of granules were used for association, the first was constituted with 60% HA and 40% TCP, and the second with 20% HA and 80% TCP.

Results demonstrated a higher stability of scaffolds with gene vectors for 60% HA/40% TCP, than for 20% HA/80%TCP formulation, because of dissolution/precipitation phenomenon of TCP more reactive phase during association process.

4) Composite matrices Ceramic/Polymer Fibers [Task T3.3]

As bone is a biological composite material made of collagen and apatite, the biomimicry stake for Biomatlante company and Inserm laboratory in the context of Gamba project was to develop a fiber-based composite matrix able:

- To improve cell adhesion in 3D, along bioinert polymer fibers and on/in osteoconductive ceramic granules.
- To embed ceramic granules in fiber mesh, which provide controlled spacing and avoid frictions between granules.
- To increase mechanical strength and allow better handleability.

To obtain such composite fiber-based scaffolds, a one-step, room temperature process was used: electrospinning. This method is available to synthesis nano or microfibers from a polymer solution thanks to high voltage potential between syringe containing solution and a metallic collector.

The chosen polymer was polylactic acid (PLA), since its hydrolytic degradation rate in vivo and bionierty constitute assets to be eligible in such medical devices.

Macroscopic granules embedded in PLA fiber matrix were the newly formulated granules described above. The next step would have been the association of the composite with the vectors to test the integrity of the transfection materials.

5) Physico-chemical assessment [Task T3.1; T3.2; T3.3; T3.4; T3.5]

Characterization methods used for quality assessment and control monitoring of our scaffolds were:

• X-Ray diffraction (XRD) to identify and quantify crystalline phases (HA/TCP).
• Infra-red spectroscopy (FTIR) to identify chemical groups and highlight undesirable molecules.
• Mercury intrusion porosimetry (MIP) to evaluate pore distribution and bulk density.
• Helium pycnometry to measure the skeletal “true” density.
• Physisorption test (BET) to measure the specific surface area (i.e. area available to proteins adsorption from body fluids invasion).
• Elemental analysis (EDX) to detect possible impurities.
• Zeta potential to evaluate electric surface charges in solutions
• Scanning Electron Microscopy (SEM) to visualize macro- microstructure and grain boundaries.

A series of adsorption-release test of proteins have also been subcontracted to expert in these fields: CIC-IT institute (Bordeaux, France) and Atlantic Bone Screen (ABS) company (St-Herblain, France).

6) Manufacturing and sterilization process [task T3.4 T3.5]

Equipment qualification, process validation for up-scaling from laboratory, sterilization efficiency according international quality standard and staff training was scheduled in order to ensure safety, efficiency and reproducibility of the device which has to be combined with the vectors and cDNA just before implantation..

To start the industrial valorization of these results, the proof of concept regarding the association of the genetic materials and the device is the first step and has still to be performed.

II. Biological evaluations: in vitro tests

1) Cell adhesion [task T3.1]

A first criterion for a dedicated hosting cells scaffold is to allow cells to spread on its surface. Human mesenchymal stromal cells were spread onto MBCP scaffolds and visualized by SEM. It obviously appeared that cells attached and spread into concavities of macropores.

2) Cell viability [task T3.1]

Cells after spreading on ceramic granules have to be able to proliferate; it will depend on the biocompatibility of biomaterials. So as to evaluate this viability, a biochemical test (MTS) was performed using murine bone cells (murine bone cells). Results highlighted a very good viability in comparison with a positive and negative control, over 90% viability.

3) Cell differentiation [Task T3.1]

When stem cells are recruited from blood or bone marrow, they adhere to the surface of ceramic granules, then proliferate and if there is a favorable environment cells differentiate into bone cells. So as to assess the osteoinduction of our materials, a reverse transcription polymerase chain reaction (RT-PCR) analysis was performed. Results shown osteogenic differentiation potential was hundred times higher when cells were spread on osteoconductive ceramic granules than along bioinert polymer fibers (without granules).

4) Active substance adsorption/release [Task T3.1]

Active substance adsorption/release quantification has been done in a comparison study between round dense granule, MBCP granule and new lower density granules. Albumin protein involved in cell adhesion was tested, as well as specifically designed Peptide 8 from ABS company and vancomycin antibiotic (CI-IT Bordeaux).

It was noticed that MBCP granules are eligible for drug delivery purpose, thanks to interconnected porosity and higher specific surface arera, allowing gradual release overtime without burst effect at short time.

Conclusion

The joint efforts of Gamba consortium partners made it possible to develop multiple potential solutions to the challenges raised by gene therapy and bone tissue engineering stakes. MBCP technology demonstrated all its assets to answer osteoconduction and osteoinduction objectives. As to new granules formulation with large internal concavity and lower density, they are highly promising in order to offer favorable environment to patient cells by acting as phosphocalcic niches, protecting cells and providing them with ions precursor of biological apatitic precipitations leading to mineralization of osteoid tissue. Composite hydrogel or fiber polymer based-scaffold have also been successfully developed thanks to one-step process. Combined properties of these inert 3D polymer meshes and highly osteopromotive ceramic granules constitute an exciting outcome obtained thanks to Gamba European project.

To go further, the association of the devices (granules, composites based on hydrogel or fibers) with the genetic materials has to be tested in vivo to validate their therapeutic potential.

WP4: Attenuation of inflammation for osteochondral defect repair by immune modulation

Lead beneficiary: NUI Galway

WP leader: Mary Murphy

Osteoarthritis (OA) is a degenerative disease that represents an enormous medical and socio-economic burden worldwide. Despite intensive research over many years, treatments for the disease remain an unmet need and there is no accepted way to repair damage due to osteoarthritis or prevent the onset of this disease. One of the major challenges to address in establishing novel therapies for OA is inflammation. Although damage to articular cartilage is considered a primary feature there are also changes to the subchondral bone and synovium. It is known that inflammation is a major driver of disease progression in OA acting in an inhibitory manner on regenerative molecular processes. In fact synovial inflammation occurs in very many patients and is a predictive factor in disease progression.

The objective of this work package was to establish optimal conditions for spatiotemporal, controlled inducible cytokine release to achieve immune modulation in order to facilitate repair of pathologies in the injured osteoarthritic joint. Interleukin 10 (IL-10) has both anti-and pro-inflammatory functions; however, the viral analog (vIL-10) is known to have an attenuating influence on inflammation. The GAMBA concept anticipated the incorporation of a gene shuttle with the coding information for vIL-10 in the upper compartment of the GAMBA modules. The proposed genetic switch was the cyclooxygenase-2 (COX-2) promoter which has been shown to respond to inflammatory signals. The goal was to implement a feed-back mechanism where inflammatory molecules released by the synovium would activate production of vIL-10 through the COX-2 promoter, thereby reducing the inflammation and silencing the signalling switch in a cyclical manner.

Task 1: Selection of optimal anti-inflammatory vector system

Plasmid DNA was cloned into COX-2 inducible promoter containing cassettes. Non-viral and adenoviral shuttle vectors containing a short (-372/+59) and long form (-1432/+59) of the COX-2 promoter were constructed for assessment of inducible expression of vIL-10. In addition they provided vectors with a reporter gene. For this task the selected reporter was secreted Metridia longa luciferase (METLuc). Initial assessment indicated successful functionality of the inducible constructs in cell lines. Following purification of the viruses, a validated method for the viral transduction of human mesenchymal stem cells (hMSCs) using lanthanum chloride was established. Furthermore, an in-house ELISA for the assessment of vIL-10 release from the cells was optimised and the ability of hMSCs to respond to optimised levels of tumour necrosis factor (TNF)-α and IL-1β as the inflammatory signals by increased expression of COX-2 established (Fig. 4.1). Despite intensive and detailed assessment it was found that the COX-2 promoter constructs were not functional at regulating vIL-10 in hMSCs whether after a primary (Fig. 4.2) or secondary inflammatory challenge: induced COX-2 expression or production of anti-inflammatory vIL-10 was not found. We also investigated whether regulated vIL-10 release could be achieved following non-viral transduction with COX-2 promoter driven vIL-10 plasmids. Although vIL-10 release at low levels was found with the constitutive CMVIL-10 plasmid following transfection of hMSCs with lipofectamine, no vIL-10 was detected with the COX-2 inducible promoter plasmids (Fig. 4.3). The use of the constitutive CMV promoter, which was observed to generate high levels of vIL-10 release from hMSCs, was selected for further in vitro mechanistic analyses and assessment of immune modulation in a mouse model of OA. In terms of alternatives to a genetic switch for controlled release of vIL-10, the pharmacological switch, doxycycline, was found to be very effective in inducing release of vIL-10 from both human and mouse MSCs transduced with a tetracycline (tet)-inducible vIL-10 virus (Fig. 4.4).

Task 2: Systematic assessment of the anti-inflammatory effect of released vIL-10

An in vitro model of inflammation using LPS activation of the monocyte cell line THP1 was established to assess the anti-inflammatory ability of vIL-10 conditioned medium (CM) from hMSCs. Release profiles for vIL-10 were established post hMSC transduction using CMVIL-10 for these mechanistic studies. It was shown that vIL-10 reproducibly reduced the levels of TNFalpha release and gene expression in THP1 cells following stimulation with LPS (Fig. 4.5).

Maintenance of cartilage integrity or promotion of a pro-chondrogenic environment is important for establishment of a reparative process in OA. Therefore, investigations with EMC were performed to assess the potential of vIL-10 in dampening inflammation in vivo using an ex vivo model with chondrogenesis of hMSCs achieved on an alginate gel modulated by CM from osteoarthritic synovial tissue (SCM). Factors secreted from the osteoarthritic synovial tissue were clearly shown to inhibit chondrogenesis by decreased gene expression for collagen II and aggrecan. Additionally, experiments were performed to assess the ability of vIL-10 to prevent inflammation.. Synovium and fat pad were obtained from patients undergoing total joint replacement with informed consent and ethical approval from local ethics committees. The following objectives were addressed: to investigate whether 1) inflammatory mediator production by osteoarthritic synovium and polarised macrophages can be modulated by treatment with vIL-10, 2) the inhibition of MSC chondrogenesis by osteoarthritic synovium is due to macrophages, and identify a role of macrophage phenotype in the inhibition of MSC chondrogenesis, and 3) whether chondrogenesis may be rescued by modulating macrophage subtype with vIL-10.

Recombinant vIL-10 was not found to reproducibly dampen inflammatory mediator production by end stage osteoarthritic synovium as assessed by quantification of pro-inflammatory IL-6, cytokine, or anti-inflammatory CCL18 [Chemokine (C-C motif) ligand 18]. The effect of SCM and vIL-10 treated SCM on collagen type 2 gene expression in hMSCs was also assessed to determine the potential of vIL-10 to modulate chondrogenesis. Although vIL-10 had a positive effect on chondrogenesis in the absence of treatment with SCM, this effect was ablated with SCM present. Again donor variability was a feature of the response to vIL-10. Levels of collagen type 2 mRNA were significantly increased in Donors 2 and 4 after v-IL-10 treatment in the presence of SCM. No effect was seen in Donor 1 but vIL-10 was inhibitory in Donor 3.

vIL-10 stimulation of M1 and M2 macrophages did not significantly alter the release and gene expression of inflammatory mediators. Human monocytes were isolated from peripheral blood and induced to the M1 phenotype by treatment with 100ng/ml LPS and 10ng/ml IFNgamma and to the M2 phenotype by exposure to 10ng/ml IL-4. Morphological analysis showed the characteristic appearance of M1 and M2 cells. M1 macrophages demonstrated characteristic expression of IL-6 and cytokine secretion with M2 macrophages showing increased expression and secretion of CCL18. Exposure to 100ng/ml vIL-10 had no effect on these parameters and also did not impact expression of TNFalpha, CD206 and IL-10 itself. Finally, the effect of M1 and M2 conditioned media on chondrogenesis of hMSCs and the impact of vIL-10 on this process was assessed. Expression of type II collagen and aggrecan was significantly inhibited by exposure to M1 CM and vIL-10 was not able to rescue this effect. M2 CM had a slight inhibitory effect on the expression of the chondrogenic factors. vIL-10 did promote expression of collagen type II mRNA but not aggrecan. Joint fat pad CM also decreased the collagen type II and aggrecan expression of MSCs.

Task 3: In vivo transfection and cytokine expression

As a strategy to achieve expression of vIL-10 in the joint, we investigated the efficacy of direct intra-articular vector injection in a small animal model. This strategy for in vivo transfection and cytokine expression required the use of a murine collagenase-induced model of osteoarthritis. CMVIL-10 was used to assess whether in vivo transduction could be achieved and an anti-inflammatory effect of released vIL-10 could be demonstrated in vivo.

At Time 0, all mice received two injections of 1U collagenase over 2 days. One week after the first injection, an equivalent amount of CMVIL-10 virus used to transduce hMSCs at MOI 100 was injected alone. All mice were humanely sacrificed at seven weeks following collagenase injection. Popliteal and inguinal lymph nodes were harvested and digested to generate a single cell suspension for analysis of T cell and macrophage populations using flow cytometry. A trend of decreased levels of CD11b+ and CD11b+Ly6C hi inflammatory monocyte levels in both the inguinal and popliteal lymph nodes was observed 6 weeks post intra-articular injection with AdIL-10. Cytokine expression in vivo was assessed following analysis of harvested serum using a multiplex ELISA assay to investigate released levels of vIL-10 following in vivo transduction, as well as other inflammatory mediators. However, available reagents were not sufficiently sensitive.

Task 4 Ex vivo transduction and immune modulation

Thereafter, in vivo assessment of AdIL-10 expressing hMSCs under the control of the CMV vector was also performed. Five groups of 8 C57BL/6 mice, 8-10 weeks old were tested. Groups were as follows: Vehicle (saline), AdIL-10, MSCs, Adnull MSCs and AdIL-10 MSCs. A dose of 20 x 104 hMSCs was injected into the treated joint, identified as the optimal cell number required for the production of 100 ng/ml vIL-10, following in vitro transduction studies. hMSCs were transduced with CMVIL-10 or Ad-Null virus at MOI of 100, using Lanthanum Chloride based transduction. Furthermore, an equivalent amount of CMVIL-10 virus used to transduce hMSCs at MOI 100 was injected alone. Blood was harvested prior to collagenase injection, 1 week post-treatment injection and again at sacrifice. Serum was isolated and frozen for cytokine analysis until required. Cytokine expression analysis, and lymph node harvest and analysis were performed as described above. Treated knee joints were harvested, fixed in 10% formalin overnight and decalcified in EDTA for 2 weeks prior to paraffin embedding for histological analysis of OA progression. The level of OA damage and degree of synovial inflammation in all treated joints was graded by three blinded reviewers. Assessment of inflammatory cell subsets was performed to investigate whether vIL-10 expression resulting from in situ transduction, or release from transduced hMSCs has an effect of inflammatory cell phenotype within the lymph nodes. Preliminary analysis indicated a decrease in the total number of CD4+ and CD8+ T cells locally in the popliteal lymph node in response to intra-articular injection of hMSCs transduced to express vIL-10. At the optimal times used for analysis of immunemodulation by vIL-10, assessment of synovium damage and osteoarthritis progression was not significantly different between groups and additional experiments will need to be performed to see if the changes that are seen in terms of the inflammatory environment result in protection from OA at later time points.

Conclusions: COX-2 inducible adenoviral vectors were not found to be functional at regulating vIL-10 in hMSCs although controlled release of the cytokine was achieved with chondrocytes in vitro. The inducible release of vIL-10 from human and mouse MSCs following doxycycline stimulation of cells that were co-transduced with AdCMVrtTA and AdTREtvIL-10 vectors, was achieved. OA synovium and infrapatellar fat pad inhibit chondrogenic differentiation of MSCs, and treatment of the synovium with vIL-10 does not counteract this effect. The negative effect of OA synovium can be addressed to the presence of M1 macrophages and the cytokines that are responsible for the negative effect of OA synovium signal act, at least partially, via the inflammatory pathways TAK1 and JAK. These results indicate that vIL-10 has the potential to impact the osteoarthritic joint pro-inflammatory environment but this effect may be limited by the level of inflammation. In vivo transfection utilising AdCMVIL-10 viral vectors served as an alternative strategy to achieve vIL-10 release and subsequent immune modulation. A trend in decreased levels of CD11b+ and CD11b+Ly6Chi inflammatory monocyte levels was achieved in both the inguinal and popliteal lymph nodes, 6 weeks post intra-articular injection with AdIL-10, suggesting modulation of inflammatory processes. In vivo immune modulation following intra-articular injection of hMSCs transduced with AdvIL-10 was also evident, with reduced CD4+ and CD8+ T cell levels detected following treatment with AdIL-10 MSCs. A significantly higher proportion of CD4+CD62L- cells in the AdvIL-10 MSC treated group compared to hMSC alone was also evident, which may suggest an increase of effector memory cells in response to this treatment. Observed trends in modulation of the development of osteoarthritic symptoms need to be addressed at a later time point, and with mouse MSCs.

WP5 Spatiotemporal control of growth factor gene delivery for cartilage repair

Lead beneficiary: EMC

WP leader: Gerjo van Osch

We have demonstrated that inflammatory factors secreted by synovium from osteoarthritis patients prevent the formation of cartilage by stem cells originating from bone marrow. Specific drugs can partially counteract this effect: they prevent the biological communication of the inflammatory factors into the cells, but the timing of administration appeared to be an important issue.

The inflammatory factors are mainly produced by certain immune-cells present in synovium: macrophages. To favor cartilage formation in a patient with osteoarthritis, changing these macrophages from producing inflammatory factors towards producing positive factors is of great importance. So far, IL10, a protein that can have such an effect was tested for this purpose. Unfortunately this was not sufficient. Further experiments using other stimulants to change the behaviour of synovial macrophages are necessary.

In order to let stem cells originating from bone marrow form cartilage under laboratory conditions, a stimulant called TGFb is generally essential. The TGFb should be present the whole time in sufficient quantities. We were not able to reach sufficiently long expression using plasmids to transfect human stem cells. However, in a model that we developed, the presence of additional TGFb appeared to be no longer necessary. Joints that are left over from slaughtered cows were used to obtain cartilage-bone biopsies: pieces of bone that are covered with cartilage on top. In this cartilage we can make defects to simulate cartilage damage. Using this model we tested the capacity of stem cells originating from bone marrow to form cartilage. Factors produced by the bone were sufficient to start cartilage formation. Initially we hypothesized that TGFb produced by the bone would be responsible for this effect. However we have proven using multiple ways to prevent TGFb from having its effect on stem cells that the bone must produce other factors that are stimulating the stem cells to form cartilage. We have not yet identified which factor or which combination of factors is responsible for this.

The positive effect of the bone was also influenced by the type of material that was used to encapsulate the cells. When we used HA-pNIPAM or alginate, the stem cells started to form cartilage, where they did this to lesser extent when we used fibrin. This effect was even stronger when we instead of culture in the laboratory, transferred this model system of cartilage-bone biopsies under the skin of mice: cartilage was formed by the stem cells in alginate and not in fibrin. The use of fibrin even resulted in unwanted bone formation, a phenomenon that is known to occur in stem cells. Alginate appeared to prevent this.

Altogether, we have developed a model system that is more representative for the situation in a joint than conventional laboratory models. This model can be used to screen cell types and/or materials for their use in cartilage regeneration. This screening is of vital importance towards the development of treatments for patients with osteoarthritis as we have found that the specific material used to encapsulate cells can have major effects on the outcome.

WP6 Spatiotemporal growth factor delivery for bone formation

Lead beneficiary: IRCCS (FORMERLY INRC OR USMI)

WP leader: Chiara Gentili/ Ranieri Cancedda

Workpackage 6 is dedicated to spatiotemporal growth factor delivery for bone formation.

One of the initial objectives was to define the temporal requirements for BMP2 exposure to obtain osteogenic differentiation. While the recombinant protein is approved for clinical use in some applications, major research is required to establish the use of its precursor, the corresponding genetic constructs have been accomplished by partner 1 (TUM), that they had constructed non-viral and adenoviral vectors for expression of TET-on BMP-2.

We have achieved inducible BMP2 expression in human MSC following Doxycycline stimulation of cells. We observed that at early passage the BMP-2 MSC increase osteogenic differentiation in vitro, but BMP-2 transfection probably interfere with the proliferation and differentiation of the MSC in culture, restricting the ability to expand cells in vitro.

Combining scaffolds and vectors

To test the efficacy of endochondral-bone formation using constructs/vectors developed in GAMBA consorsortium, in WP6 we evaluated with different type of cells, human bone marrow stromal cells (BMSCs) and human osteoblasts (OB) the capacity to form bone in vivo. We loaded the scaffolds that we received from Biomatlante partner, only with cells and we implanted them in mice to evaluate the efficacy of bone formation of the scaffold plus and minus cells. Human and sheep BMSCs were isolated from adult bone marrow and cultured in vitro. Human OB were harvested from bioptic pieces of large bones by several mechanical and enzymatic digestions and cultured according to (Tortelli et al. Tissue Eng Part A. 2009 Sep;15(9):2373-83).

We used two micro/macroporous biphasic calcium phosphate ceramics (MBCP) with different composition; MBCP A, composed of 60% of hydroxyapatite and 40% of tricalcium phosphate beta and MBCP +, composed of 20% of hydroxyapatite and 80% of tricalcium phosphate beta. To evaluate the effect of the different calcium phosphate ceramics composition on ectopic bone formation, we used hMSCs and hOB and positive control cells sheep MSC, we seeded 2 x 106 cells at passage 1 into MBCP (A and +) scaffolds. Immunodeficient mice (CD-1 nu/nu; Charles River, Calco, Italy) were anesthetized and four subcutaneous pockets created. Scaffolds of each of the calcium phosphate ceramics were implanted for 1, 4 and 8 weeks. At the end of time points, animals were sacrificed and implants were harvested for histological analysis to access bone formation. As required by the Italian Ministry of Health, all animals were maintained in accordance with the standards of the Federation of European Laboratory Animal Science Associations (FELASA).

The results shown that using different MBCP scaffolds loaded with no transfect BMSC a good bone formation occur. Moreover, we analyzed several histological sections to compare bone size and dimension for both scaffolds and results that hBMSCs deposit more bone extracellular matrix in the scaffolds made with MBCP plus granules (Fig.6.1).

Histomorphometric analysis was performed to quantify bone and blood vessel amounts. The results shown an overall bone formation in both scaffolds loaded with hMSC. We also observed a significantly larger amount of blood vessels in the condition with more bone formed. The induction of large and mature bone associated with vast presence of organized blood vessel structures might be related not only to different microporosity grade but mainly to Ca2+ ion flux product of CaP dissolution/re-precipitation events occurring in bone microenvironment.

From the in vitro studies, previous described, we observed that BMP-2 transfection interfere with the proliferation and differentiation of the MSC in culture, restricting the ability to expand cells in vitro. For these reasons, we have not obtained the correct number of cells to load on MBCP scaffolds, and performed the in vivo studies using BMP-2/MSC combined with MBCP scaffolds. Moreover, in collaboration with WP1 (OZB, TUM) we performed in vivo transfection studies with lipoplexes/GFP cDNA and Microporous Biphasic Calcium Phosphate (MBCP) granules provided by BIM (WP3). We used DOGTOR lipid, identified in previous experiments as a favourable transfection reagent for hMSC and we combined this lipid complex and MBCPs scaffolds to perform an in vivo 3D scaffold transfection. These constructs should be useful for following directly the in vivo transfection of the cells recruited from the scaffolds.

Testing the efficacy to repair defects in small animal models

Since biphasic calcium phosphate (BCP) granules are known to be osteoconductive, INSERM, Biomatlante, TUM and OZB have evaluated the osteoinductive effect of MBCP+™ micro-macroporous substitutes using heat-induced model of rabbit osteonecrosis in femoral epiphysis sites.

Therefore, after assessing the therapeutic osteogenic potential of these BCP granules, the ultimate objective of this work was to combine MBCP+™ granule therapy with specifically designed gene vectors (Dogtor, OZbioscience, Marseille, France) whose plasmid is coding for BMP2 growth factor.

Critical size defects (5–6 mm in diameter and 8 mm in length) were induced in femoral epiphyses of 36 New Zealand rabbits. A thermocouple, which was controlled by a temperature probe, was heated to 80°C. Rotation of the thermocouple was performed in order to improve homogeneity of the thermal treatment, which lasted for 45 seconds. Following implantation of loaded/unloaded bioceramics scaffolds with vector/plasmid, the animals were confined for 3, 6, and 12 weeks (Figure 6.2).

Results demonstrated the necrosis model induced by focal heating insults was effective as a very highly significant difference was observed between empty-necrosed site and ceramic filled sites. A highly osteoinductive potential of MBCP+™ granules in osteonecrosed sites was proven systematically by histological analysis (Figure 3). The addition of Dogtor/plasmid system to MBCP+ granules shown at least as good results in terms of new bone volume but not statistically significant differences were highlighted. These results are then promising since optimization of vector/plasmid concentration on granules and improvement of the association protocol could be done thanks to further investigations.

WP7 Large animal models for OA

Lead beneficiary: ARI

WP Leader: Mauro Alini

The aim of this Workpackage was to perform a proof of concept of one or more modules created by the previous WPs in a large animal model, the goat. Although the isolation and expansion of the equivalent cell populations developed in the workpackage 3-5 was established for the goat model, the large animal pre-clinical trial (WP-7) was not performed as the therapeutic solutions envisioned could not be fully proven in preliminary small animal studies. Therefore, the consortium could not ethically and scientifically approve the trial. However, small animal study was performed by ARI to demonstrate the biocompatibility of the biomimetic hyaluronan hydrogel. The rabbit model used was relevant for the GAMBA project aiming to repair both cartilage and bone tissues. It has the advantage to be a simpler in vivo model, useful for pre-screening biomaterials and tissue engineering solutions in comparison to the more clinically relevant but complicated and ethically more demanding goat model.

The in vivo model consisted in an osteochrondral defect on the weight-bearing surface of the medial femoral condyle of a rabbit with a diameter of 2.7 mm and a depth of 4 mm and created using a drill bit. The study was approved by authorized ethical commission. In this defect a thermoresponsive hydrogel was injected or left empty respectively. During the study radiographs were taken before (entry test) and immediately after surgery. Weights were controlled pre operatively, 3 days and 7 days after surgery. The rabbits were euthanized 1 or 12 weeks after surgery. Post mortem radiographs were taken. All rabbits recovered well from surgery and all clinical exams are within normal limits for the duration of the entire study. During the study no high burden for the animals was detected as shown with the individual scorings during the whole study duration. Histological findings indicated that the biomimetic hydrogel was biocompatible and could therefore be used as a delivery carrier in vivo.

WP8: Linking GAMBA to society through science-society dialogues on chances, risks and ethical aspects

Lead beneficiary: SCID

WP Leader: Katharina Zöller

Workpackage 8: Societal Views: Patient and Citizen Panels on GAMBA

What is a lay panel?

In a lay panel, a group of 12-25 interested participants develop a joint statement on a socially relevant topic. Participants receive information understandable to lay people, and inter¬view experts some of whom they can choose themselves. They are supported by a team of professional, neutral facilitators. The term “lay panel“ is derived from “citizens' panel“, which is a form of participation inspired by the methods of “citizens jury” and consensus conferences. The goal is to develop a joint lay opinion in the form of a “lay report“.

The GAMBA lay panels took place between May 2011 and July 2012 on 3,5 resp. 4 days per panel (two weekends with an interval of three weeks) in Munich/Germany, Galway/Ireland and Zollikerberg near Zurich/Switzerland with altogether 71 participants aged 19 to 82. The participants of the citizens' panels were partly recruited random¬ly. In this way we hoped to avoid recruiting only participants who are already (very) actively involved in societal debates. Another advantage of recruiting at random is that participants from differing social backgrounds can be reached.

The lay panels are a type of „microcosm“, reflecting a smaller image of social reality. The whole group’ assessment, and not – as in a survey – the sum of the uninformed individuals' opinions, is leading to the results, as they are based on intensive discussions and negotiations.

One central element in the procedure of the lay panels is a wide variety of expert input. It sets out to inform the participants on the whole range of subject-specific insights and assess¬ments. This ensures that opportunities as well as uncertainties and risks are discussed, and ethical questions are raised.

This innovative form of public participation in science in professionally facilitated lay panels enables the participants to take a stand on the complex subject matter of GAMBA (gene and stem cell therapies). They were able to advise the scientists, the EU as our sponsor, research politics and other agents on societal issues on GAMBA in a knowledgeable way.

Overview of the lay panel process:

In the lay panels, participants were asked to assess the GAMBA field of research from their various perspectives as patients or interested citizens. To prepare them for this, they first received an introduction to GAMBA as a field of research: in the run-up to the panels, participants received the “Manual” and “Compendium” brochures which had been written in layman’s terms by the science journalist, Beatrice Lugger, in collaboration with the project team. The panels listened to presentations (on osteoarthritis, the proposed mode of operation of the GAMBA approach, possible risks and the ethical aspects of gene and stem cell therapies) and questioned self-selected experts directly in a hearing. Equipped with this information, they then discussed the opportunities, possible risks and ethical aspects of GAMBA, and after an extensive discussion in breakout groups and in plenary they drew up modules for submitting their opinions (lay report in German and English: http://www.wissenschaftsdialog.de/index.php/download).

Participants prepared their “Lay Statements” in two sessions (of two days each) on the issues relating to GAMBA. The panels’ first session served to “empower” participants in the subject and methodology:

• Day 1: After participants had been given an opportunity to get to know each other and were provided with an outline of the schedule, they listened to an introductory presentation on osteoarthritis, were given an overview of the GAMBA research project, put their questions to the speakers and discussed the issues with them.
• Day 2: On the second day, participants considered GAMBA in depth with the local re-search teams. The lay participants also listened to presentations on the possible risks related to the GAMBA field of research and on the ethical aspects. The session concluded with participants selecting experts for a hearing on the second weekend and they were given the opportunity to extend their knowledge of the individual aspects of GAMBA in terms of ethical and social factors between the two sessions through the creation of “ambassadorships”, i.e. individual participants adopting an issue to pursue on behalf of the panel.

The second session (three weeks later) was wholly geared towards the development of assessments, opinions and the suggested wording of the lay report:

• Day 3: On the third day, those participants who had prepared a “ambassadorship” presented or discussed their results with their fellow panellists. Then the hearing with the experts selected by the participants took place.
• Day 4: The fourth and last day was used for an in-depth discussion and for the assessment of the GAMBA field of research. The panellists prepared modules of text covering the opportunities, risks and ethical aspects of GAMBA which were then composed by the ScienceDialogue team and – after the panels - fed back to the participants for comments and approval. Two elected spokespersons presented the recommendations at the final event in each country.

Each day ended with a “snapshot review”, i.e. a brief appraisal of the respective day. The facilitation team was flexible in addressing the needs of the individual groups, accepted criticism and suggestions and made changes. Therefore, even though the process is comparable overall, there may be minor local variations.

Summary of the recommendations of lay participants in the GAMBA panels: see attachment

Quality criteria for scientific lay panels

The following quality criteria are important for public participation processes:

Clear mandate: From the start it is clearly defined what will be done with the results and who the intended audience is comprised of. These addressees should commit to making a qualified public statement and include the lay recommendations in their work wherever possible.

Methodical empowerment of participants: This is supported by an inclusive, experienced and appropriate method of facilitation, which is committed to upholding the quality criteria (below). The facilitators empower the self-confidence and the assessment skills of the participants, allowing a discussion between laypersons and experts at eye level.

Qualified input of information: This must be ensured by a qualified information base (brochures and other sources of information) which is balanced and understandable for laypersons, by expert presentations from various perspectives, as well as influence on the choice of experts and possibilities for own research.

Transparency: It is important to provide information about panel objectives including creative leeways and limits. Furthermore, a continuous visualization and document¬ation of the process supports the processing of information and the achievement of results.

Outcome openness and impartiality: Participants must be able to influence the infor-mation process through the selection of thematic aspects and experts as well as their own research; outcomes are generated as the sole responsibility of the participants.

WP9: Disseminating and exploiting GAMBA

Lead beneficiary: TUM

WP Leader: Christian Plank, Martina Anton

Dissemination of knowledge has been on several levels:

Internal dissemination

- regularly scheduled progress meetings, phone conferences, electronic communication
- electronic communication, creating and continuously updating common knowledge bases
- the webpage contains a secured area with access restricted to partners for exchange and dissemination of information amongst partners.
- the webpage contains a list of project associated publications of partners and others.
- lab visits of students in order to learn new techniques

External dissemination:

External dissemination has been on several levels: to the interested lay-public and to the scientific community.

- a webpage was created to allow for information on GAMBA
- press releases: on general issues like granting of the project by the EC, on selected results
- publications in non-scientific journals to attract maximal attention to GAMBA outputs and impact on the therapies availability and safety for patients suffering from arthritis
- publications in scientific journals
- presence on international meetings: presentation of scientific results
- organisation of symposia and satellite meetings on international congresses
- citizen and patient panels were part of the GAMBA WP8
- the presentation of the project objectives, results and impacts in workshop sessions open for professionals
- effective communication with the media (television, radio and press) has been established in order to better inform the public, be more visible and increase citizen’s awareness and interest in arthritis’ research. Numerous press articles and broadcasts made the wider public aware of the project.

Scientific community dissemination (including clinicians).

A public progress report has been posted on the webpage at regular intervals. As part of the GAMBA project, 20 papers were accepted for publication in scientific journals and 9 master and PhD theses were completed by students participating in the project. Partners also attended international meetings to communicate their findings.

Educational material has been prepared and made available to students to strengthen their expertise in nanomedicine (WP8).

Three commercial gene delivery products for tissue engineering were launched and one patent application is under way.

WP10 Management Activities

Lead Beneficiary: TUM

Work package leader: Martina Anton, Christian Plank

Objective:

The objective of this WP was to ensure the following:

that all budgetary actions are performed correctly and according to the rules and regulations established by the European Commission and in the consortium agreement; that the received funds are correctly distributed and accounted for, including independent auditing; that the work and tasks are completed in time, within budget, and satisfy high-quality requirements; that reporting is done on a periodic basis, in the most efficient and pragmatic way, according to Commission guidelines to provide all consortium members with all important and impacting information that can influence the projects’ outcome; to implement an authority ensuring that knowledge and innovation are properly managed and results exploitation promoted; to prepare measures for patent application

The consortium started with the Kick-off meeting organized by the coordinating partner TUM in Steinebach Wörthsee, Germany (near Munich) on 16.-17.09.2010. We had a total of 23 participants with every partner sending at least the PI and one more representative. There the steering committee as highest body of decision making within GAMBA was established with one representative of each partner. The steering committee was chaired by Martina Anton as coordinator. Christian Plank (TUM) was appointed as exploitation manager.

The six-month meeting was held on 24.-26.02.2011 in Davos, Switzerland. This meeting was attended by the PIs and additional investigators of all partners. Additionally, the project officer Dr. Johan Veiga-Benesch and the EU appointed project technical advisor Dr. Roxana Piticescu attended the meeting. Additionally the following phone conferences took place: 20.12.2011 between TUM and NUI Galway;25.01.2012 between ARI, TUM, OZB, BIM, INSERM The midterm GAMBA meeting was held in Nantes, FR: 26.-27.03. 2012. The 24 months meeting was held in Vienna, AT, 08.-09.09.2012 on the occasion of th TERMIS conference. The 30 months meeting was held in Genova, IT, 11.-12.2013 and the final meeting was held in Munich, DE, 11.-12.07.2013. All meetings were attended by at least one representative of each partner and all meetings included a steering committee meeting as well. The final meeting in Munich was also attended by the Project Officer Dr. Jaime Ibanez de Elejalde and the project technical advisor Dr. Roxana Piticescu. All meetings were very effective in terms of scientific exchange and in terms of implementing the work plan.

An exploitation plan had been compiled by the exploitation manager as well as the final plan of use and dissemination of foreground. These plans were approved by all partners and were subsequently submitted to the Commission.

Ethical issues: No experiments were performed without obtaining permissions from the appropriate regulatory bodies beforehand. Ethical approvals had been submitted to the EC and updates have been obtained and communicated during regular reporting.

The most challenging task was, like in all EU projects, the reporting business. Accordingly, this was the most time-consuming task to WP10. All reports were submitted in a timely or close to timely manner.

Potential Impact:

Impact:

The GAMBA project has addressed several key topics of the call [NMP-2009-2.3-1] as well as of the general work programme summarized below.

Cell based therapies: Mesenchymal stem cells have been activated by genes in the materials to repair damaged tissues.

Osteoarthritis/Osteoporosis: GAMBA technologies have been validated in models of osteoarthritis. Results will be widely applicable in tissue repair and have implications for future application in osteoporotic bone fractures.

Use of bioactive molecules coupled to engineered biomaterials: The bioactive molecules are gene vectors. The biomaterials are engineered to allow for spatiotemporal control.

Advanced multidisciplinary approaches: The consortium has been a multidisciplinary team uniting biomaterials science, gene therapy approaches, medical sciences, immunology, stem cell research, translational research, social sciences and economic exploitation.

Local tissue repair: Our materials were designed for tissue repair.

Inhibition of inflammation: The multicomponent system comprises gene vectors responding to inflammatory molecules with the expression of anti-inflammatory molecules.

Materials which can match and exchange stimuli with the natural biological environment and stimulate healing: The system was designed to adopt anatomical structures, to respond to biological and physical stimuli and to induce healing.

Bio-inspired materials based on natural or synthetic biomimetic gels and polymers: The materials used here entirely conformed to this requirement.

Radical innovation in state of the art biomaterials: Implementation of spatiotemporal control of specific cellular actions to orchestrate tissue regeneration.

Materials with electromagnetic properties: Superparamagnetic nanoparticles were included to allow for spatiotemporal control.

Potential for medical applications: Potential for future medical applications is clearly evident and has been discussed with affected patients and the general public.

Gender and age-related: Patient panels with mainly female and elderly patients and workshops with female employees: current situation, career expectations and demands.

Within GAMBA, materials have been developed to result in biomedical implants having characteristics close to those of natural tissues. The innovative approach within GAMBA is expected to contribute to an increased competitiveness of the European biomaterial and biomedical industry by giving rise to patentable innovations to be exploited by the existing networks of the SME and academic Partners.

The GAMBA project was complementary to other previous and on-going Europe-wide and national research programmes. Notably, Partner ARI is represented by an international network of more than 10’000 surgeons specialized in treatment of trauma and disorders of the musculoskeletal system. Erasmus MC participates in all large national consortia in the field of regenerative medicine and osteoarthritis (Dutch Platform for Tissue Engineering, Translational Excellence in Regenerative Medicine; “Smart-Mix”, BioMedical Materials programme, Top Institute Pharma). In addition Erasmus MC is coordinator of a Marie Curie Fellowship program on bone regeneration. Ranieri Cancedda’s laboratory has been involved in many EU sponsored projects, such as Angioscaff, Purstem, Join(ed)T, and ERISTO IV. The Regenerative Medicine Institute (REMEDI) is a biomedical research centre with a central focus on the development of novel therapies for human diseases using adult stem cell therapy and gene therapy. GAMBA researchers have been involved in additional EU-sponsored projects including Purstem ADIPOA, BioStem and ANGIOSCAFF, ERISTO. INSERM, EMC, ARI and IRCCS were partners of cost project (Namabio, Franco Rustichelli coordination). More specifically INSERM and Biomatlante are partners in bone regenerative medicine (REBORNE project, 7th framework program HEALTH-2009-1.4.2 of the European Commission on Regenerative Bone defects using New biomedical Engineering approaches)

Among the GAMBA partners have been two SME’s which can commercialize materials and reagents emerging from the GAMBA project. OZ Biosciences, France, is active in the reagents business. Biomatlante, France, is a well-known manufacturer of implantable biomaterials for regenerative medicine. The whole consortium and the companies in particular contribute to reinforcing the European competitiveness and innovation as summarized in the tables below.

Direct economic benefits:

• Decrease time and cost of novel treatments (matrix-based; activated on command and demand) developments for cartilage and bone disease supported by the high efficient technological package and associated processes developed by the GAMBA Partners,
• Improve European competitiveness in the osteoarthritis field,
• Additional patents and competitive positioning of GAMBA Partners on the technologies and models to be developed,
• Better management of ageing population suffering from osteoarthritis (novel treatment strategies).

Indirect economic benefits:

• New treatments for osteoarthritis, Bone regeneration using bone substitutes
• Significant reduction of European Health care costs associated to osteoarthritis,
• Improved knowledge and competitiveness of Europe in osteoarthritis, bone regeneration using bone substitutes
• Pro-active campaign for acceptance of technology by the general public

GAMBA outputs:

• New engineered multifunctional gene vectors,
• Novel transfection strategies using non-viral or adenoviral vectors with feedback response elements driving transgene expression
• New biomimetic thermo-responsive hyaluronan hydrogel
• Multiphasic association of Biomimetic Calcium Phosphate granules
• Innovative granules to enhance bone regeneration
• New Scaffolds for Drug Delivery Systems
• New in vitro models for evaluation of therapies to repair osteochondral defects
• New dialogue tool (Panel concept) in include societal/ethical aspects/values into research

Short-term innovation:

• New gene vector carriers,
• Innovative matrices formed by MSCs cells, biomaterials and gene vectors
• Spatiotemporal control of chondrogenesis and osteogenesis of MSCs embedded in gene-activated materials and development of a model that has properties close to natural tissue
• Patentable design of hollow granules for better osteoconduction
• Novel strategies for immune modulation in a controlled spatiotemporal manner resulting in attenuation of in vivo joint repair for osteochondral defects and osteoarthritis

Long-term innovation:

• Innovative matrices providing remedies and innovative model to study and treat osteoarthritis disorder.
• Enlargement of GAMBA technologies spectrum of action to other cartilage and bone disorders e.g. osteoporosis, dental implants.
• Extension of GAMBA to wound healing (skin defects), healing of tendon etc.
• General application of GAMBA to tissue engineering by selection of appropriate promoters and cDNAs

This is connected to significant market perspectives. The osteoarthritis market is predicted to grow steadily, and will reach $7 billion by 2015*.

*From the report: Commercial Insight: Osteoarthritis - Market sees steady growth (2006)

Furthermore, GAMBA may have a pronounced societal impact in a long-term perspective. In Europe, around 6 per cent of the European population suffer from frequent knee pain and radiographic osteoarthritis. It has been estimated that around 35 to 40 million European suffer from osteoarthritis in 2007. It is estimated that around 25 per cent of people with age 60 and above suffer from disability due to osteoarthritis. The economic impact of musculoskeletal diseases in industrialised countries varies from 1 to 2.5% of Gross Domestic Product, with reference to local situations and to different social policies. As osteoarthritis (OA) is the most common joint disorder, it absorbs most of the resources among musculoskeletal diseases. GAMBA proposes new therapeutic approaches and treatments for osteoarthritis thus making possible for the suffering population to significantly enhance their quality of life and to reduce morbidity-related costs.

GAMBA may have an impact on standards as well. With the long-term goal of exploiting results of the project in clinical application, European regulatory authorities will be engaged in the future. The novelty of the involved technologies will require adaptation of current regulatory provisions.

GAMBA has for the entire funding period or parts of it generated a number of positions: 12 persons (full time or part time) were hired specifically for the GAMBA project at the different partners, thus increasing work force at the partners institutions. Additionally to persons paid by the GAMBA project scientific as well as technical or administrative personnel was involved in GAMBA. Scientists included master students and internships that contributed essentially to the project although typically not being paid positions. Thus the GAMBA project contributed to scientific education at the partner universities as well. In total three doctoral thesis and six master theses have been produced in the context of GAMBA. Additionally to education of university students GAMBA researchers of TUM presented the GAMBA project in a life video presentation broadcasted to higher eduction students in the “Deutsches Museum, München” Munich during the “Nanoday” 09.02.2011.

Furthermore the GAMBA manual and compendium to the GAMBA manual are well suited as science education manuals explaining the integral topics of gene therapy, adult stem cells, nano-particles, regulation of gene expression.

The GAMBA project has given rise to an extensive collaboration of 3 biotech companies, 2 of them having been members of the GAMBA consortium (OZB and Biomatlante). The third company, ethris GmbH in Munich, Germany, is a spin-off of TUM and works on bone regeneration with messenger RNA encoding bone-inducing factors. It is intended to continue this collaboration beyond the GAMBA project.

Wider societal implications:

In order to actively involve lay people a whole WP (WP8) was dedicated to engaging civil society in the GAMBA project. For this purpose citizen and patient panels were performed in three countries (Germany, Ireland and Switzerland). The aim of these panels was to inform participants in lay language about the aims, chances and risks of GAMBA. To allow an open neutral discussion, moderators were involved. Science Dialogue (SCID), who are social scientists and professional moderators, but were not involved in the natural/medical sciences/engineering was a partner to the consortium.

With the help of SCID participants produced a lay report on these subjects with the main emphasis on ethics, chances and risks of the approach. These reports were published in the original languages (German and English) and also were translated.

Furthermore these lay reports were communicated to policy makers, by sending the print versions and publishing on the internet.

In order to increase public awareness about the GAMBA concept in the wider community and to address relevant societal issues citizen and patient panels were integral part of the GAMBA project. With the help of partner SCID in WP8 lay persons (citzens and patients) were involved and encouraged in getting to know, discuss and actually give input into GAMBA.

As can be seen in WP9 extensive scientific as well as non-scientific dissemination of GAMBA concept and results have been achieved.

TUM and SCID generated press releases related to GAMBA and especially to the panels.

SCID published the lay reports on the internet as well as sent print versions to the EC and policy makers. Additionally SCID sent the final lay-reports to the participants of the panels, thus using them as multipliers to other lay persons. With these measures, they contributed to maximizing the awareness of the public of the GAMBA project.

The Regenerative Medicine Institute at NUI Galway has a core outreach ethos and aims

• to increase public awareness of research carried out at the centre
• to increase awareness and interest in science among young people and to encourage them to consider further education or a career in this field
• to engage the public, including patient groups and carers, in discussions of future applications and ethical implications of contemporary scientific research

This effort is necessary to create the ability to become prominent as a resource for stem cell research comment and analysis in the Irish media. GAMBA’s significant effort in addressing societal issues as a core element of the project is instrumental in achieving these aims both locally in all partner countries but also in a broader European context. Dissemination and real outreach/engagement efforts targeted to patients and the general public have ensured this. This effort will ensure maximum awareness of the contribution of GAMBA to advances in nanomedicine, incorporating stem cell and gene therapies as well smart biomaterials, leading to more effective therapies in the future. By engaging with patients and the public so effectively throughout the course of the project, GAMBA has also increased awareness of these novel therapies and promoted ultimate acceptance of novel therapies that may result from GAMBA output.

NUI Galway contributed to achieving awareness as follows:

NUI Galway built on the GAMBA press release by TUM in Munich and adapted the information for a release of the launch of the project on the NUI Galway website. An invitation to the work-package leader, Mary Murphy, to participate in a radio Interview post press release was received as a result.

NUI Galway also had significant exposure associated with WP8 outreach activities: specifically, dissemination of GAMBA occurred as a result of activities in WP8 and the implementation of Task 8.4 “Patient and citizen panel in Ireland”.

In response to a NUI Galway press release, published on the NUIG website, to promote the Patient Panel in an effort to initiate recruitment, many media hits ensued. These included a radio interview and subsequent daily promotion of GAMBA by Galway Bay FM for over 7 days. Additionally, seven online media hits and 3 articles in the local print media were noted. Additional effort concentrated on liaising with Arthritis Ireland and their access to member details and dissemination of leaflets through local hospitals, doctor’s offices and clinics, libraries, pharmacies and health stores.

In addition to media exposure for the Patient Panel reported in the reporting period covering the first 18 months of the project, an additional 3 newspaper articles focusing on the GAMBA panel held in February and March 2012.

As there are no comprehensive voter databases in Ireland due to the ability of citizens to opt out of disseminated registration databases, we initiated a significant effort to advertise the Citizen Panel in order to recruit participants for the panels held in May and June 2012. Efforts included dissemination through letters to the local county council, secondary schools, 20 Active Retirement Groups, Galway branch of the Green Political Party, Literary and Debating Society at the NUI Galway, local libraries, Galway City Community Forum all students and staff at NUI Galway, volunteer organisations, a half page advertisement in the local newspaper (Galway Advertiser) and a press release on the NUI Galway website.

Seven media hits resulted from this effort to advertise the citizen panel in the period to the end of May 2012. The launch of the combined Citizen/Patient panel report was held on Nov 2, 2012 and further elicited a number of reports online and in the print media.

Recently, one of the GAMBA patient panellist was featured on a TV piece on stem cells and a new initiative recently launched at NUI Galway on production of stem cells for clinical trials. All media exposure is listed in Deliverable 10.2 (T9.3: Public relations campaign).

Gender issues:

A Gender work shop was held during the Genova meeting 11.-12.02.2013 with female participant from the following partners: TUM, NUI Galway, IRCCS, EMC and SCID. These researchers also contributed to the gender workshop to emphasise gender equity in GAMBA organized by SCID.

To prepare the gender work shop a questionair prepared by SCID had been previously sent to all female members of the consortium and the WP leader presented the outcome of the survey.

An excerpt of the results of the gender work shop was presented to the whole consortium during the following GAMBA meeting, highlighting the roles, requirements and wishes of women in the scientific research world.

Dissemination and Exploitation

Dissemination of knowledge has been on several levels:

Internal dissemination:

• regularly scheduled progress meetings, phone conferences, electronic communication
• electronic communication, creating and continuously updating common knowledge bases
• the webpage contains a secured area with access restricted to partners for exchange and dissemination of information amongst partners.
• the webpage contains a list of project associated publications of partners and others.
• lab visits of students in order to learn new techniques
• External dissemination
• External dissemination has been on several levels: to the interested lay-public and to the scientific community.
• a webpage was created to allow for information on GAMBA
• press releases: on general issues like granting of the project by the EC, on selected results
• publications in non-scientific journals to attract maximal attention to GAMBA outputs and impact on the therapies availability and safety for patients suffering from arthritis
• publications in scientific journals
• presence on international meetings: presentation of scientific results
• organisation of symposia and satellite meetings on international congresses
• citizen and patient panels were part of the GAMBA WP8
• the presentation of the project objectives, results and impacts in workshop sessions open for professionals
• effective communication with the media (television, radio and press) has been established in order to better inform the public, be more visible and increase citizen’s awareness and interest in arthritis’ research

Scientific community dissemination (including clinicians). A public progress report has been posted on the webpage at regular intervals. Numerous manuscripts have been published in scientific journals, several are under review and some are in preparation. Partners also attended international meetings to communicate their findings.

Educational material has been prepared and made available to students to strengthen their expertise in nanomedicine (WP8).

The following peer reviewed papers have been published by members of the consortium during the above mentioned funding period:

Cédric Sapet, et al. 2012. Magnetic nanoparticles enhance adenovirus transduction in vitro and in vivo. Pharm Res. 29(5): 1203-18. (Date of publication: 01.05.2012)

Christian Plank, et al. 2012. Gene Activated Matrices for Bone and Cartilage Regeneration in Arthritis GAMBA – an EU-Funded Project. European Journal of Nanomedicine 4(1): 17-32 (Date of publication: 20.05.2012)

Matteo D’Este et al. 2012. Single step synthesis and characterization of thermoresponsive hyaluronan hydrogels. Carbohydrate Polymers. 90(3): 1378-85. (Date of publication: 13.07.2012)

Thomas Miramond et al. 2012. In vivo Comparative study of two injectable/moldable Calcium phosphate Bioceramics. Key Engineering Materials 529-530: 291-5. (Date of publication: 29.11.2012)

Thomas Miramond et al 2012. Composite bioceramics/polymer electrospun scaffolds for regenerative medicine. Key Engineering Materials 529-530: 441-6. (Date of publication: 29.11.2012)

Guy Daculsi et al. 2012. Calcium Phosphate Bioceramic scaffold for bone tissue engineering. Key Engineering Materials 529-530: 19-23. (Date of publication: 29.11.2012)

Matteo D’Este et al. 2013. Hydrogels in calcium phosphate moldable and injectable bone substitutes: Sticky excipients or advanced 3-D carriers?. Acta Biomaterialia 9(3): 5421-5430. (Date of publication: 01.03.2013)

Rui C. Pereira et al. 2013. Dual effect of platelet lysate on human articular cartilage: a maintenance of chondrogenic potential and a transient pro-inflammatory activity followed by an inflammation resolution. Tissue Eng Part A. 19 (11-12): 1476-1488. (Date of publication: 30.01.2013)

Markus Berninger et al. 2013. Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots. Journal of Visualized Experiments 75: e4423. (Date of publication: 21.05.2013)

Sapet et al. 2013. 3D-fection: cell transfection within 3D scaffolds and hydrogels. Therapeutic Delivery. 4 (6): 673-685. (Date of publication: 01.06.2013)

Zöller, Katharina: Laien diskutieren und bewerten Wissenschaft: Partizipation bei Wissenschaftsthemen. Erfahrungen und Qualitätsanforderungen. In: UMID 2/2013. 67-74.

Zöller, K., science Dialogue. 2013. GAMBA Manual and Compendium, In Press Bioceramics Development and Applications. BDA 203, Journal of the International Society for Ceramic in Medicine, ISCM.

Thomas Miramond et al. Osteopromotion of Biphasic Calcium Phosphate granules in critical size defects after osteonecrosis induced by focal heating insults. IRBM (accepted for publication 24.07.2013)

M.L. de Vries – van Melle, R. Narcisi, N. Kops, J.L.M. Koevoet, P.K. Bos, J.M. Murphy, J.A.N. Verhaar, P.M. van der Kraan, G.J.V.M. van Osch. Chondrogenesis of mesenchymal stem cells in an osteochondral environment is mediated by the subchondral bone. (Accepted for publication in Tissue Engineering Part A – 27.08.2013)

Barry F, Murphy M.. 2013. Mesenchymal stem cells in joint disease. Nat Rev Rheumatol. [epub ahead of print Jul 23, 2103; DOI 10.1038/nrrheum.2013.109]

Zöller, Katharina: Science and the Lay Perspective: Lay Peoples’ involvement in assessing Tissue Engineering Issues. 2013. Accepted for publication at the journal Tissue Engineering Part C.

The following manuscripts are under peer-review:

De Vries-van Melle et al. European Cells and Materials

Van Beuningen, de Vries-van Melle et al. Tissue Engineering A

Fahy, de Vries-van Melle et al. Osteoarthritis and Cartilage

The following book chapter has been published:

G. Daculsi, T. Miramond. Calcium phosphate derived biomaterials. Encylopedia of Biophysics (Roberts, GCK, ed.). 01.03.2013: 206-211.

The following doctoral theses have been produced:

T. Miramond. Développement de matrices céramiques et composites pour l’ingénierie tissulaire osseuse. (Ph.D thesis defence, date: 27.11.2012; INSERM/BIM)

M.L. de Vries-van Melle. In vitro models for cell-based cartilage regeneration. (Anticipated defence: spring 2014; EMC)

Pereira, Rui. Insights into the role of biochemical factors and extracellular matrix environment during chondrocyte culture: relevance on articular cartilage repair strategies. Ph.D thesis in preparation (IRCCS)

The following master theses have been produced:

Stefan Sandker: Osteochondral culture model. (2010, Technical Medicine University Twente/EMC)

Rene van der Bel. A highly adaptable osteochondral culture model towards stimulating microfracturing in vitro. (2011 Technical Medicine University Twente/EMC)

Lizette Utomo. Optimization of the preparation, cell encapsulation and analysis of hydrogels (2011 Technical Medicine University Twente/EMC)

Johannes Lehmann: Modulating Synovial Macrophage phenotypes to improve stem cell based cartilage repair. 2012 (EMC)

Maria Tihaya: Comparing the ability of three hydrogel cell carriers to support repair of osteochondral defects by mesenchymal stem cells in an in vitro model 2012 (EMC).

M. Hillreiner. Evaluation von Gen-aktivierten Matrices zur Kontrolle der Genexpression. (Evaluation of gene-activated matrices for the control of gene expression). (2012; TUM)

Patents:

A patent application for hollow granules by BIM and INSERM is underway.

List of Websites:

http://www.gamba-project.eu/

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